Glycated hemoglobin (in particular, “HbA1c”) present in blood cells serves as a significant indicator in the diagnosis, therapy, etc. of diabetes because the concentration thereof reflects the patient's past history of blood glucose levels. In general, HbA1c is measured by an immunoassay, HPLC, or the like.
However, in whole blood or blood cells, not only glycated hemoglobin (glycated Hb) as an analyte but also various glycation products such as glycated amines (e.g., glycated albumins, glycated peptides, and glycated amino acids) are present as non-analytes. Thus, there has been a problem in that the glycation products (the non-analytes) other than the analyte also are measured, so that the measured value becomes greater than the true value or a false positive result is obtained
In order to solve such a problem, in the field of immunoassay, the following methods have been proposed, for example. That is, a method has been proposed in which, before causing a main reaction between glycated Hb as an analyte and an antibody against the analyte, a complex of a non-analyte with an antibody against the non-analyte is formed so that the structure of the non-analyte is changed to that causing no influence on the immunoreaction of the glycated Hb (see Patent Document 1, for example), and a method has been proposed in which a non-analyte is separated and removed from a sample by B/F separation. On the other hand, in the field of HPLC, a method has been proposed in which two HPLC columns are provided for one sample so that a non-analyte is removed from the sample in the first column and an analyte is separated and analyzed in the second column, for example.
However, the above-described immunoassays have a problem in that they require a high cost and besides, an antigen-antibody reaction other than the main reaction needs to be performed further, which complicates the environment setting of the reaction system and causes the measurement to take a long time. Furthermore, with regard to the immunoassay using the B/F separation, it is obvious that the operation is complicated. Still further, because an antibody only acts on a substance (an antigen) that exhibits specificity to the antibody, when the type of a non-analyte contained in the sample is unknown or many types of non-analytes are contained in the sample, it is difficult to remove the influence of the non-analyte(s) sufficiently. Moreover, according to the above-described HPLC, a cost increase cannot be avoided because it uses two columns. Besides, the separation of the non-analyte takes a long time, so that, when the improvement in the measurement accuracy is intended, there is a limit to the reduction in the measurement period.
On the other hand, in recent years, enzymatic methods utilizing a redox reaction have been applied widely to the measurement of various glycated proteins including glycated Hb, and attempts have been made to put these enzymatic methods into practical use. Specifically, the measurement is carried out in the following manner.
First, blood cells are hemolyzed to prepare a sample. To the thus-obtained hemolyzed sample, a protease is added so that a degradation product of glycated Hb is generated. Then, a fructosyl amino acid oxidase (hereinafter referred to as “FAOD”) further is added to the degradation product so that the FAOD acts on a glycation site of the glycated Hb to cause a redox reaction, thereby generating hydrogen peroxide. The amount of the hydrogen peroxide corresponds to the amount of the glycated protein. Then, a peroxidase (hereinafter referred to as “POD”) and a substrate that develops color by oxidation are added to the reaction solution so that the substrate develops color through the enzyme reaction. The amount of the hydrogen peroxide can be determined by measuring the color developed. As a result, the amount of the glycated protein in the blood cells can be determined.
However, as described above, such an enzymatic method also has a problem in that the measured value becomes greater than the true value owing to a glycation product as a non-analyte contained in the sample. Therefore, in order to solve this problem, the applicant of the present invention proposed the following enzymatic methods (e.g., Patent Document 2).
That is, the applicant of the present invention proposed, as a first method, a method of measuring glycated Hb as an analyte by adding a FAOD (a degradation FAOD) exhibiting a low reactivity to the glycated Hb to a sample beforehand to treat a glycation product as a non-analyte and then treating a degradation product of the glycated Hb obtained through a protease treatment with a FAOD (a measurement FAOD) exhibiting a high reactivity to the glycated Hb.
The applicant of the present invention also proposed, as a second method, a method of measuring glycated Hb as an analyte by pretreating a sample with a small amount of a FAOD (a degradation FAOD) exhibiting a high reactivity to the glycated Hb beforehand, then treating the sample with a protease, and adding the same FAOD (a measurement FAOD) again to the sample. In this second method, the reason why the amount of the degradation FAOD to be added is small, more specifically, the reason why the ratio of the pretreatment FAOD to the measurement FAOD is small, is that this allows the glycated Hb to be prevented from reacting with the FAOD during the pretreatment in terms of the kinetics. Moreover, in the second method, the protease is added not only to degrade the glycated Hb but also to deactivate the degradation FAOD added first. This is because, if the degradation FAOD added first remains, the degradation product of the glycated Hb generated by adding the protease reacts with the remaining FAOD.    [Patent Document 1] JP 2000-180439 A    [Patent Document 2] WO 03/033729